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E2F4-RB and E2F4-p107 complexes suppress gene expression by transforming growth factor beta through E2F binding sites
1Department of Pharmacology, Duke University Medical Center, Durham, NC 27708, USA.
Abstract:
Transforming growth factor beta (TGF-beta) causes growth arrest in most cell types. TGF-beta induces hypophosphorylation of retinoblastoma susceptibility gene 1 product (RB), which sequesters E2F factors needed for progression into S phase of the cell cycle, thereby leading to cell cycle arrest at G1. It is possible, however, that the E2F-RB complex induced by TGF-beta may bind to E2F sites and suppress expression of specific genes whose promoters contain E2F binding sites. We show here that TGF-beta treatment of HaCaT cells induced the formation of E2F4-RB and E2F4-p107 complexes, which are capable of binding to E2F sites. Disruption of their binding to DNA with mutation in the E2F sites did not change the expression from promoters of E2F1, B-myb, or HsORC1 genes in cycling HaCaT cells. However, the same mutation stimulated 5- to 6-fold higher expression from all three promoters in cells treated with TGF-beta. These results suggest that E2F binding sites play an essential role in the transcription repression of these genes under TGF-beta treatment. Consistent with their repression of TGF-beta-induced gene expression, introduction of E2F sites into the promoter of cyclin-dependent kinase inhibitor p15(INK4B) gene effectively inhibited its induction by TGF-beta. Experiments utilizing Gal4-RB and Gal4-p107 chimeric constructs demonstrated that either RB or p107 could directly repress TGF-beta induction of p15(INK4B) gene when tethered to p15(INK4B) promoter through Gal4 DNA binding sites. Therefore, E2F functions to bring RB and p107 to E2F sites and represses gene expression by TGF-beta. These results define a specific function for E2F4-RB and E2F4-p107 complexes in gene repression under TGF-beta treatment, which may constitute an integral part of the TGF-beta-induced growth arrest program.
Insights
Transforming growth factor beta (TGF-beta) halts cell growth by forming E2F-RB complexes. These complexes bind E2F sites, repressing specific gene expression and contributing to TGF-beta
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Transforming growth factor beta (TGF-beta) is a key regulator of cell growth, typically inducing cell cycle arrest at the G1 phase.
- This arrest is mediated by the hypophosphorylation of the retinoblastoma susceptibility gene 1 product (RB), which sequesters E2F transcription factors, preventing cell cycle progression.
- The precise mechanisms by which TGF-beta represses gene expression, particularly concerning the role of E2F-RB complexes, remain to be fully elucidated.
Purpose of the Study:
- To investigate the role of E2F-RB and E2F-p107 complexes in TGF-beta-induced gene repression.
- To determine if E2F binding sites are essential for the transcriptional repression of specific genes under TGF-beta treatment.
- To elucidate the functional mechanism of E2F-RB/p107 complexes in mediating TGF-beta's growth arrest program.
Main Methods:
- Treatment of HaCaT cells with TGF-beta.
- Analysis of E2F4-RB and E2F4-p107 complex formation and DNA binding.
- Site-directed mutagenesis of E2F binding sites in gene promoters (E2F1, B-myb, HsORC1, p15(INK4B)).
- Reporter gene assays to measure promoter activity.
- Chimeric construct experiments using Gal4-RB and Gal4-p107.
Main Results:
- TGF-beta treatment induced the formation of E2F4-RB and E2F4-p107 complexes capable of binding to E2F sites.
- Mutating E2F binding sites significantly increased the expression of E2F1, B-myb, and HsORC1 promoters in TGF-beta-treated cells, indicating repression.
- Introduction of E2F sites into the p15(INK4B) promoter repressed its TGF-beta-induced expression, and tethering RB or p107 to this promoter via Gal4 sites also inhibited induction.
- These findings demonstrate that E2F binding sites are crucial for TGF-beta-mediated transcriptional repression.
Conclusions:
- E2F-RB and E2F-p107 complexes play a critical role in repressing the transcription of specific genes in response to TGF-beta.
- E2F binding sites are essential for mediating this transcriptional repression, acting as docking sites for these repressor complexes.
- The findings define a specific mechanism for gene repression by E2F-RB/p107 complexes, integral to the TGF-beta-induced cell growth arrest program.